Antibiotic resistance is now seen as one of the major challenges facing modern medicine. Given the shortage of novel antibiotics, a number of alternative approaches are being investigated, including the use of bacteriophages as therapeutic agents (Barrow & Soothill, Trends in Microbiology (1997), 5, 268-271; Dixon B, The Lancet Infectious Diseases (2004), 4, 186; Hausler T, Viruses vs. Superbugs: A Solution to the Antibiotics Crisis? (2006) MacMillan, New York; Matsuzaki et al, Journal of Infection and Chemotherapy (2005), 11, 211-219.
Bacteriophages (often known simply as “phages”) are viruses that grow within bacteria. The name translates as “eaters of bacteria” and reflects the fact that as they grow most bacteriophages kill the bacterial host as the next generation of bacteriophages is released. Early work with bacteriophages was hindered by many factors, one of which was the widespread belief that there was only one type of bacteriophage, a non-specific virus that killed all bacteria. In fact, the host range of bacteriophages (the spectrum of bacteria they are capable of infecting) is often very specific. This specificity may be considered a therapeutic strength as populations of bacteriophages can be selected to specifically eliminate only the target bacterial species.
Despite the therapeutic advantages afforded by the host specificity of bacteriophages, this characteristic has the disadvantage that it can be difficult to achieve breadth of coverage of target strains. For this reason, there has been interest in finding combinations of bacteriophages having broad targeting capability in relation to particular types of bacterial infection (see for example Pirsi, The Lancet (2000) 355, 1418). This has now been achieved with the development of a mixture of six bacteriophages targeting Pseudomonas aeruginosa, which has completed veterinary field trials and is now in human clinical trials (Soothill et al, Lancet Infectious Diseases (2004) 4, 544-545). The challenge now is to develop regimens which optimise the delivery of such therapies.
Bacteriophages and antibiotic therapy have previously been used together in Eastern Europe (see for example Bradbury, The Lancet (February 2004) 363, 624-625), but without specific reporting of synergistic effects. Indeed, there have been suggestions that antibiotics can have adverse effects on use of bacteriophage therapy since bacteriophages use bacterial metabolism to replicate and this is inhibited by antibiotics (Payne and Janssen, Clinical Pharmacokinetics (2002) 42, 315-325).
More recently, bacteriophages have been shown to produce benefits where mixed pathogenic bacteria grow in a biofilm (Soothill et al, 2005, PCT patent application WO2005009451). In this application benefit was shown with respect to subsequent antibiotic treatment of heterologous bacterial infections, apparently by disruption of the biofilm following bacteriophage treatment.
Biofilm formation is now known to be a characteristic of many important pathogenic bacteria contributing to increased resistance to antibiotics. Such bio films may comprise more than one type of bacterium supported and surrounded by an excreted extracellular matrix and assist bacteria to colonize surfaces from marine reefs to tooth enamel. Biofilms allow bacteria to attach to surfaces and to attain population densities which would otherwise be unsupportable. They impart increased resistance to not only antibiotics but many environmental stresses including toxins such as heavy metals, bleaches and other cleaning agents. It was previously thought that contribution of biofilm formation to antibiotic resistance was primarily a physical process arising from limitation of diffusion, but more recent evidence has shown that some bio films appear to have specific abilities to trap antibiotics (Mah et al., Nature (2003) 426, 306-310). It is known that bacteria within biofilms can be 100 to 1000 times more resistant to antibiotics than the same strain of bacteria growing in single-celled (“planktonic”) forms. This increased resistance means that bacteria that are apparently sensitive to antibiotics in a laboratory test may be resistant to therapy in a clinical setting. Even if some are cleared, biofilms may provide resistant reservoirs permitting rapid colonization once antibiotics are no longer present. It is clear therefore that biofilms are major factors in many human diseases.
As noted above, greater beneficial effects have been observed with the subsequent use of antibiotics against mixed infections following the use of a therapeutic bacteriophage preparation against Pseudomonas aeruginosa, and it has been proposed that this is due to the destruction of Pseudomonas aeruginosa as the key species maintaining the bio film (Soothill et al, 2005, PCT patent application WO2005009451), which results in loss of biofilm integrity and thus exposure of bacteria to conventional antibiotics.
The teaching of PCT patent application WO2005009451 is against the use of antibiotics that are specifically active against the same bacterial species as that targeted by bacteriophage. The examples cited refer to the use of Synulox (amoxicillin and clavulanic acid) and/or Canaural ear drops (containing diethanolamine fusidate, framycetin sulphate, nystatin and prednisolone). Both of these preparations contain only antibiotics that are not effective against Pseudomonas aeruginosa (Krogh et al, Nordisk Veterinaer Medicin (1975) 27, 285-295; Kucers A, in Kucers et al (eds), The Use of Antibiotics: A Clinical Review of Antibacterial, Antifungal and Antiviral Drugs, Fifth edition (1997), Butterworth-Heinemann, Oxford; Rawal, Journal of Antimicrobial Chemotherapy (1987) 20, 537-540). In particular, while aminoglycoside antibiotics as a class are effective against Pseudomonas aeruginosa, Framycetin is of very limited efficacy. Kucers notes that “Nearly all the medically important Gram-negative aerobic bacteria are sensitive” (to Neomycin, Framycetin and Paromomycin) “with the exception of Pseudomonas aeruginosa”, while the same author states that “Pseudomonas aeruginosa is co-amoxiclav resistant, citing the work of Comber et al, in Rolinson & Watson Augmentin (eds) (1980), Excerpta Medica, Amsterdam, p. 19. Co-amoxiclav is defined in the online 52nd edition of the British National Formulary (www.bnf.org) as “a mixture of amoxicillin (as the trihydrate or as the sodium salt) and clavulanic acid (as potassium clavulanate), equating to the veterinary drug Synulox. Thus, PCT patent application WO2005009451 would not indicate the use of antibiotics targeting Pseudomonas in any combination with bacteriophages but rather the use of antibiotics specifically targeting co-infecting bacteria.
Another mechanism has been identified recently by which bacteriophages can increase the sensitivity of bacteria to antibiotics to which they are resistant (Hagens et al, Microbial Drug Resistance (2006), 12, 164-168). This involves active bacteriophage metabolism, and is suggested to involve the formation of pores in the bacterial membrane. However, this teaches that “resensitization of pathogens resistant to a particular antibiotic can be achieved in the presence of phage in vivo” based around the use of “a combination treatment with antibiotics and filamentous phage”. Thus this relates to a non-heritable characteristic which is exerted only in the presence of bacteriophage, which relies on the simultaneous use of both bacteriophages and antibiotics, and which appears to be specific to filamentous bacteriophages which form pores in the bacterial membrane. This is thus distinct from the inventions claimed herein, which induce heritable changes that persist even when actively replicating bacteriophage is not present.